Human neutrophil gelatinase is a component of specific granules.

Previous investigators have proposed that gelatinase, a metalloproteinase found in neutrophils, is stored in a novel secretory compartment distinct from the two major granule populations, azurophilic and specific. To locate this proteinase in human neutrophils we reacted the cells for peroxidase and then applied monospecific polyclonal antibodies to human neutrophil gelatinase to immunolabel ultrathin frozen sections using an immunogold technique. Gelatinase was localized in a population of peroxidase-negative granules. Double-labeling experiments using antibodies against lactoferrin, a marker for specific granules, and gelatinase demonstrated colocalization of the two antigens in 80% of the specific granules. However, some granules immunostained with only the lactoferrin or gelatinase antibody. Similar techniques were used to examine precursor cells from bone marrow. In myelocytes both gelatinase and lactoferrin were present in large developing specific granules; however, some mature specific granules contained only lactoferrin. Thus, it is possible that lactoferrin synthesis begins earlier than gelatinase synthesis and that overlapping synthesis and segregation occurs during the myelocyte stage. These findings suggest that the main storage compartment of gelatinase is within the peroxidase-negative specific granules.

Detection of gastric pepsin in middle ear fluid of children with otitis media.

OBJECTIVE: We sought to confirm the finding of pepsin/pepsinogen in the middle ear fluid of children with otitis media in a larger sample size using a sensitive and specific pepsin assay. STUDY DESIGN AND SETTING: We evaluated 152 children (225 ear samples) in a prospective study at a tertiary care children's hospital. The presence of pepsin in middle ear aspirates was determined using enzymatic assay. RESULTS: Of the patients, 14.4 percent (22 of 152) had detectable pepsin activity in one or both of the ear samples with no pepsin activity detected in control serum. Average pepsin concentration in the samples was 96.6 +/- 170.8 ng/ml, ranging from 13 to 687 ng/ml. Pepsin concentration in the middle ear of children younger than 1.0 year was significantly higher than in older age groups. CONCLUSION AND SIGNIFICANCE: Results indicate that pepsin/pepsinogen is present in the middle ears of children with otitis media, although not at the high rate previously reported. Gastric reflux may be one causative factor in the pathogenesis of otitis media.

Pepsin assay: a marker for reflux in pediatric glue ear.

OBJECTIVE: To evaluate if analysis of pepsin/pepsinogen in middle ear effusions can be considered a diagnostic marker for laryngopharyngeal reflux (LPR) in children with otitis media with effusion (OME). MATERIAL AND METHODS: Ambulatory 24-hour dual-probe pH monitoring was carried out on 31 children with OME. Middle ear effusions were collected from 17 children during myringotomy. Total pepsin/pepsinogen concentrations in effusions were measured by ELISA using antipepsin antibody. RESULTS: Dual-probe pH monitoring showed that 22/31 (71%) of the studied children had significant LPR. The concentrations of pepsin/pepsinogen in middle ear effusions, ranged from 0.085 to 5.02 microg/ml, were found to be up to 4.5 to 231.44 times higher than the serum levels. There was a significant positive correlation between the level of pepsin/pepsinogen assayed in the effusions of the 17 children and the number of pharyngeal reflux episodes measured by pH monitoring. CONCLUSIONS: Control of LPR may be an essential component in the successful management of OME in pediatric patients. Pepsin/pepsinogen analysis in effusions of children, using ELISA, can be considered a reliable marker for assessment of reflux in children with OME.

[The functional status of the human stomach in the course of the experiment with antiorthostatic hypokinesia of 4 months duration].

The paper deals with type of changes in gastroduodenal organs associated with hypersecretion of the stomach induced by simulation of some effects of microgravity. Subjects in the investigation were 13 males volunteered for a 4-month bedrest study. Biochemical changes in blood, urine and gastric juices were analyzed in comparison with ultrasonic images of the gastroduodenum organs and data of stomach and duodenum endoscopy. The investigations showed increased blood and urine levels of pepsinogen indicative of gastric hypersecretion characterized by more intensive secretion of gastric juices, lowered pH, elevated pyloric tone, activation of duodenal secretion and cholepoiesis. Gastric hypersecretion was concomittant to the development of plethora in the abdominal veins. The highest gastric secretion was observed after two months of bed rest. Further bed rest extension was marked by fading of the signs of hypersecretion by the stomach and appearance of some signs of chronic stress. Our results expand the knowledge of changes in the gastroduodenal organs during bed rest, space flights and other conditions provocative of the hypersecretory transformation of the stomach.

Pepsinogen identification in the middle ear fluid of children with otitis media with effusion.

OBJECTIVES: This study aims to investigate the presence and concentration of pepsin/pepsinogen in middle ear fluid and to discuss the potential mechanisms involved in the pathogenesis of this condition. PATIENTS AND METHODS: A total of 33 children (21 boys, 12 girls; mean age 5.7±2.4 years; range 3 to 13 years) diagnosed with otitis media with effusion and scheduled for operation were enrolled into the study. Fluids aspirated from the middle ear were assessed for the presence of pepsinogen and albumin and blood samples were drawn simultaneously for comparison. RESULTS: Mean pepsinogen concentration was statistically significantly higher in middle ear fluids compared with serum samples (262.4 ng/mL [range: 211.7 ng/mL - 301.1 ng/mL] versus 102.6 ng/mL [range: 80.7 ng/mL - 134.5 ng/mL], respectively) (p<0.001). On the other hand, mean albumin concentration was significantly lower (1.1 g/dL [range: 0.01 g/dL - 9.5 g/dL] versus 5.8 g/dL [range: 0.9 - 9.5 g/dL], respectively) (p<0.001). The highest pepsinogen concentration was detected in patients with purulent effusion (275.3 ng/mL). CONCLUSION: Our findings support the theory of gastro-esophageal reflux related pepsinogen transition to the middle ear and indicate that pepsinogen may a reliable biochemical marker for the assessment of gastro-esophageal reflux.

Further purification and some properties of a gelatin-specific proteinase of human leucocytes.

A latent gelatin-specific proteinase (gelatinase) was isolated from the crude extract of human leukocytes. The enzyme was purified about 180-fold (7040 units/mg) with an overall yield of 23%. The isolated protein migrated as a single band on sodium dodecyl sulfate polyacrylamide-gel electrophoresis. Its mobility was unaffected by reducing agent. The protein band corresponded to approx. 90-94 kDa. Gelatinase activity was strongly inhibited by chelating agents, such as EDTA and 1,10-phenanthroline. This inhibition was reversed by Zn2+ and Co2+; other metal ions were less or not at all effective in reversing the inhibition. Moreover, Co2+ stimulated gelatinase activity. These results indicate that Zn2+ and/or Co2+ are essential for the activity of this gelatinase.

Loss of gelatinase in elicited peritoneal neutrophils of rats.

In previous studies on the respiratory burst of neutrophils, it was observed that the small, water-soluble quinone, duroquinone, activated the respiratory burst in rat peritoneal neutrophils but not in human blood neutrophils. Here we report studies which indicate that a selective diminution of certain granule enzymes, gelatinase and azocaseinase but not elastase, occurs during elicitation in the rat. Neutrophils circulating in rat blood have about twice the amount of gelatinase and azocaseinase activity as compared to elicited peritoneal cells, and the respiratory burst of circulating cells is stimulated to only about 20% by duroquinone. The significance of these results is considered in terms of activation of the neutrophil respiratory burst.

Expression of interstitial collagenase, 92-kDa gelatinase, and tissue inhibitor of metalloproteinases-1 in granuloma annulare and necrobiosis lipoidica diabeticorum.

Granuloma annular (GA) and necrobiosis lipoidica diabeticorum (NLD) are disorders characterized by granulomatous inflammation and degenerative changes in collagen and elastic fibers. Because these disorders have often been described as being associated with altered extracellular matrix deposition, we studied the in situ expression of interstitial collagenase, 92-kDa gelatinase, and tissue inhibitor of metalloproteinases (TIMP)-1. Twelve lesions each of GA and NLD of different histopathologic types and durations were examined. Interstitial collagenase mRNA was seen in histiocyte-like cells in one-third of the cases of both diseases, typically in younger lesions. In GA, collagenase mRNA was only detected in lesions of the palisading type. Signal for 92-kDa gelatinase mRNA was observed in eosinophils, which were present in low numbers in five of 12 GA and three of 12 NLD samples. The signal for this enzyme and the presence of eosinophils did not correlate with the age of lesion. TIMP-1 mRNA was consistently expressed by histiocyte-like cells in both disorders. In GA, TIMP-1 mRNA was detected at the outer edge of the palisading granulomas, but in NLD, inhibitor expression was seen in the perivascular and periadnexal accumulation of inflammatory cells. Our data indicate that collagenase and TIMP are expressed early in these disorders and that these proteins may contribute to stromal remodeling associated with necrobiotic lesions. Our results further indicate that the localization of TIMP-1 production may provide a distinction between the two disorders, whereas metalloproteinase expression is not sufficiently specific to aid in the differential diagnosis of GA and NLD.

Separation of fibronectin from a plasma gelatinase using immobilized metal affinity chromatography.

Conventional preparations of plasma fibronectin are known to contain a co-purifying gelatinase [1986, J. Biol. Chem. 261, 4363-4366], but so far useful methods to remove the protease have not been available. In this study a number of different methods were tested in order to achieve separation of the two proteins. Immobilized metal affinity chromatography was found to be efficient for this purpose, and a convenient procedure to separate the two proteins under nondenaturing conditions on chelating Sepharose charged with Co2+, Ni2+, or Zn2+ is described. An alternative method employing pH gradient elution of an Fe3+ gel also resolved fibronectin from the gelatinase. The Fe3+ gel bound both proteins at pH 6.0 but not at pH 7.4, suggesting that the two proteins were phosphorylated. The described procedures will now allow studies of the functions of fibronectin in the absence of the contaminating protease.

Effects of neutral proteases from human leukocytes on structure and biological properties of human factor VIII.

Human factor VIII was purified from cryoprecipitate and incubated for up to 24 hours with four neutral proteases of human blood leukocytes, namely, with elastase-like protease (ELP), chymotrypsin-like protease (CLP), collagenase and gelatinase. Electrophoretic patterns showed a reproducible sequence of degradation of factor VIII and of its 230,000 molecular weight subunit by ELP and CLP. Intermediate products were similar but those resulting from exhaustive proteolysis by ELP and CLP differed distinctly from each other. Procoagulant activity of factor VIII was rapidly and completely destroyed by ELP and CLP before visible electrophoretic changes would be detected. No increase in this activity was observed prior to its destruction. Von Willebrand factor (ristocetin cofactor) activity was considerably more resistant to ELP and CLP and declined in rough relation to degradation of highly aggregated forms of factor VIII. ELP and CLP produced a pronounced progressive increase in the Laurell reaction antigen. Normal human plasma showed a high potency to inhibit ELP and CLP. Large doses of these enzymes (300 microgram per ml) produced in the plasma medium only a moderate fall in factor VIII procoagulant activity. Collagenase and gelatinase did neither degrade factor VIII nor change its biological properties.

Inhibition by nonsteroidal anti-inflammatory drugs of superoxide production and granule enzyme release by polymorphonuclear leukocytes stimulated with immune complexes or formyl-methionyl-leucyl-phenylalanine.

The effects of nonsteroidal anti-inflammatory agents on superoxide production and granule enzyme release by human polymorphonuclear leukocytes stimulated with either formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe] or immune complexes were investigated. Cytochrome c reduction and the release of lysozyme, beta-glucuronidase, myeloperoxidase and gelatinase were measured. Auranofin, phenylbutazone, sulfasalazine and the phospholipase A2 inhibitor, 4-bromophenacyl bromide, strongly inhibited these responses in fMet-Leu-Phe stimulated cells, at concentrations below 50 microM. Indomethacin, piroxicam, mefenamic acid, primaquine and quinacrine at 50-250 microM were inhibitory. Up to 1 mM ibuprofen and chloroquine inhibited superoxide production but had little effect on degranulation. With cells stimulated by IgG aggregates (immune complexes), up to 1 mM ibuprofen, mefenamic acid and piroxicam did not inhibit either response. Indomethacin, phenylbutazone, sulfasalazine and primaquine inhibited, but considerably higher concentrations were required than with fMet-Leu-Phe. Quinacrine inhibited superoxide production equally well with both stimuli but inhibited enzyme release only with fMet-Leu-Phe. Only auranofin, 4-bromophenacyl bromide, and the weakly effective chloroquine exerted approximately the same effect with both stimuli. D-Penicillamine did not affect enzyme release with either stimulus and interfered in the superoxide assay. Gelatinase release induced by fMet-Leu-Phe was affected to the same extent, or slightly more, than release of the other granule enzymes. With immune complexes, there was only modest inhibition of gelatinase release by any of the drugs at 250-1000 microM. Our results reinforce previous observations that many anti-inflammatory drugs affect neutrophil functions, but their effects vary with stimulus. The relative insensitivity of immune complex-induced responses to most of the drugs must be taken into account when considering their mode of action.

Variation of total pepsin activity in sera with age and under different stimulations of gastric acid secretion.

The total pepsin activity in sera was measured in 660 outpatients endoscopically proven to be free of gastroduodenal lesions. A small but significant increase of total pepsin activity with age in females older than 50 years was observed. Total serum pepsin activity increased after stimulation of gastric acid secretion. This increase was small after a meal and vagal stimulation by sham feeding, but higher after maximal stimulation with Pentagastrin. Its amount, which is in general rather small and probably not relevant for clinical evaluation of gastric acid secretion, depends on the proportion of cases with high gastric secretory capacity. This might clarify the discrepancy in the results obtained by previous authors.

The activation of human neutrophil gelatinase.

To study the mechanisms of activation of human neutrophil gelatinase, the enzyme has been purified using a combination of chromatography on a DEAE-Sephacel and a gelatin-peptide-Sepharose column. On reducing SDS-polyacrylamide-gel electrophoresis the purified gelatinase ran as a single band of about 94,000 Da, and had a specific activity of 5624.4 units/mg of enzyme protein. When latent gelatinase was treated with trypsin, cathepsin G, neutrophil elastase, HgCl2 or urea, its activity was enhanced and the enzyme was processed and converted into species of the lower molecular mass. Upon activation, the protein band of 94,000 Da of reduced latent gelatinase underwent a decrease of about 6,000-12,000 Da. Formation of the species of lower molecular mass during urea activation could be blocked by the addition of EDTA.

Activation of latent human granulocyte gelatinase by rat mast cell protease.

Rat peritoneal mast cell extract contains an activator of latent human granulocyte gelatinase. The activator has been partially purified and characterized. It shows a similarity to rat mast cell chymase in several properties including its molecular weight, substrate specificity and sensitivity to inhibitors. The activation of latent gelatinase with rat mast cell protease is dependent on protease concentration, incubation time and is mediated through the catalytic site of the activator. The significance of mast cell protease in the regulation of collagenolytic enzymes is discussed.

Matrix degrading proteinases from human granulocytes: type I, II, III collagenase, gelatinase and type IV, V-collagenase. A survey of recent findings and inhibition by gamma-anticollagenase.

Three human matrix degrading leukocyte proteinases, type I collagenase, gelatinase and a new type IV collagenase were isolated in latent and active form. Activation of all three latent enzymes could be achieved by treatment with either organomercurials or with trypsin. In addition the 90 kDa latent type I-collagenase could be activated by disulfides, while a newly discovered 70 kDa latent form could be activated with organomercurials or with trypsin. The active type I collagenase was inhibited by gamma-anticollagenase from human serum (and the leukocyte type I collagenase inhibitor, while the newly found type IV collagenase was inhibited only partially. The complexes formed from gamma-anticollagenase with type I collagenase, i. e. latent enzyme, are not reactive site associated complexes. The binding is not of a substrate-like and competitive manner. After inhibition of the enzyme though inactive against its natural substrates it is still hydrolyzing the synthetic low molecular weight octapeptide DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH.

Gastric anti-ulcer activity of silymarin, a lipoxygenase inhibitor, in rats.

Oral treatment with silymarin was found to be effective in the prevention of gastric ulceration induced by cold-restraint stress, in rats. Statistically significant ulcer index values with respect to the control group, were observed. In 6 h pyloric-ligated animals silymarin showed a significant reduction in the number and severity of the ulcers; however, it did not alter the gastric secretion volume or acidity although histamine concentration was significantly decreased. In absolute ethanol-induced ulcers, treatment with silymarin 1 or 2 h before the anti-ulcerogenic agent, did not prevent the formation of gastric lesions. Furthermore, the hexosamine content was decreased significantly, but the total protein output was enhanced, showing similar values to those with the standard drug, carbenoxolone. These results suggest that the anti-ulcerogenic effect of silymarin could be related to its inhibitory mechanism of enzymatic peroxidation by the lipoxygenase pathway, avoiding leukotriene synthesis.

Vomit identification by a pepsin assay using a fibrin blue-agarose gel plate.

Reported is a simple and reliable method for identifying the presence of gastric fluid in forensic samples by an assay that reveals the pepsin activity. These samples are usually vomit found at the scene of a crime, either in fresh form or as a dried stain on clothing. The pepsin within the sample is assayed for its proteolytic activity which is revealed in a fibrin blue-agarose gel plate, as a result of an enzymatic reactivity that takes the form of a concentric, blue, translucent ring around the tested sample. Apart from being able to determine the pepsin content of fresh or recent forensic samples, this method has also achieved positive reactions in aged gastric fluid stains that were kept at room temperature. No body fluids other than the gastric fluid and no proteolytic enzymes other than pepsin show a positive reaction with the use of this method. This method has an additional advantage, in that the enzymatic activity seen on the gel plate can be photographed and the gel plate, on drying, can also be preserved as evidence.

Serum pepsin activity as a parameter of gastric acid secretion.

Serum pepsin activity was measured in 359 patients with various gastric diseases, the serum protein themselves serving as substrate. In 332 out of 359 patients, BAO and PAO, and in 173 of these patients, pepsin activity, were determined in gastric juices. In 102 patients, serum pepsin activity was measured before, and 45 and 90 minutes after, pentagastrin stimulation. In 71 out of these 102 patients, pepsinogen I was measured simultaneously by RIA. There was good correlation between stimulated serum pepsin activity and maximal pepsin output (r = 0.63), as well as BAO (r = 0.67) and PAO (r = 0.77). The higher the PAO, the greater the increase in pepsin activity in the sera after pentagastrin stimulation. The increase in pepsinogen I under stimulation was smaller in the sera than pepsin activity. There was a slightly better correlation of PAO with stimulated serum pepsin activity than with pepsinogen I (r = 0.76 versus 0.62). The measurement of pentagastrin-stimulated pepsin activity in sera, as a very simple and inexpensive method, permits the screening, with an overall accuracy of more than 75%, of achlorhydrics (stimulated pepsin activity less than 30 micrograms thyrosine/24 hours/ml serum), acid secretors (greater than 80 micrograms thyrosine/24 hours/ml serum) and hypersecretors (PAO greater than 35 mmol HCl/hours, greater than 120 micrograms thyrosine/24 hours/ml serum).

Changes in total pepsin activity and pepsinogen I in human sera under stimulation and inhibition of gastric acid secretion.

The levels of total pepsin activity and pepsinogen I in sera were first studied in 10 healthy volunteers undergoing stimulation of gastric acid secretion at random on 4 different days with a) a maximum dose (6 micrograms/kg) of pentagastrin, b) ranitidine injection 5 minutes before maximal pentagastrin stimulation, c) ranitidine injection 1 hr before maximal pentagastrin stimulation, or d) sham feeding. In a further 10 healthy volunteers the levels of total pepsin activity and pepsinogen I were studied over 5 days by short-term oral intake of ranitidine. The total pepsin activity and pepsinogen I increased significantly in the sera after maximum-dose pentagastrin, but not after sham feeding. The increase could be completely prevented by ranitidine given only 1 hour before pentagastrin stimulation. The total pepsin activity and pepsinogen I decreased significantly after brief oral intake of ranitidine. It can be concluded that the measurement of total pepsin activity and pepsinogen I in the sera could serve as a rough parameter for testing the effectiveness of potential agents inhibiting gastric secretion.

Method for determining serum pepsinogen concentration, using pepsin standards and ultraviolet absorbance.

A simplification of the traditional hemoglobin methods for determining serum pepsinogen concentration was developed. In this method, 10% trichloroacetic acid solution was added to control samples, and hemoglobin substrate was added to controls and active enzyme samples; standards and samples were incubated for 18 hours, the proteins in the active tubes were precipitated with trichloroacetic acid and removed by filtration, and the absorbances of the supernatant of each standard and sample at 280 nm were measured. The major differences between this method and other methods for determining pepsinogen values are that the preacidification of serum with hydrochloric acid was eliminated, the incubation period was reduced to 18 hours (down from 24 hours), the relative pepsinogen concentration was determined by measuring the concentration of hydrolysis products, using ultraviolet, rather than visible absorbance, and a pepsin standard curve was used to determine the serum pepsinogen concentration. Comparison of freshly prepared pepsinogen and pepsin standard curves indicated that the pepsinogen preparations were slightly more active than the pepsin preparations (on a weight-to-weight basis) on the same substrate. Pepsin standards are used because they are more stable than pepsinogen standards. Three linear standard curve ranges were used: O 10 to 100, 50 to 300, and 100 to 500 ng of pepsinogen/ml of serum. The use of pepsin standard curves permits some variability of the incubation conditions without altering the results. For best results, the hemoglobin substrate solution should be prepared daily. This method may be useful in diagnosing ostertagiasis.